**3.2.2.3 Effect of ultrasounds on ethanol fermentation**

In all HRTs, significant higher ethanol productions in the ultrasound-assisted fermentation process than in the control fermentation process were recorded (p<0.05). When the HRT was 12 h, the ethanol concentration without ultrasonic treatment was 9.87 g L-1 and it was significant lower by 2.85 g L-1 than the production in the process stimulated with low intensity ultrasounds (p<0.05) (Fig. 9). Lactose consumption was only 62.1%, but application of ultrasound increased it to 69.7% (p<0.05) (Fig. 10). The best results were obtained with the longest HRT of 36 h. Ethanol concentration increased to the value of 26.30 g L-1 when the culture has been sonicated, while in the fermentation process without ultrasound irradiation was only 23.60 g L-1 (p<0.05), (Fig. 9). Lactose consumption was as high as 98.9% in ultrasound-assisted fermentation unit and was significant higher by 6.5% than the consumption in the reactor without ultrasonic irradiation (p<0.05) (Fig. 10). High ethanol production and lactose consumption were observed with shortening HRT to 24 h. *S. cerevisiae* stimulated with low intensity ultrasound produced 24.85 g ethanol L-1, while the lactose consumption was 95.6% (Fig. 9–10). In the control fermentation unit there was 21.79 g L-1 and 89.5%, respectively. The differences were statistically significant (p<0.05). Under the HRT of 36 h, in the fermentation process with ultrasound irradiation the maximum ethanol yield of 0.532 g g-1 lactose was observed, whereas using biocatalyst *S. cerevisiae* without ultrasound exposure gave the result as 0.511 g g-1 (Fig. 11) (p<0.05). Shortening the HRT to 24 h allowed remaining high ethanol yield of 0.520 g g-1 with sonicated *S. cerevisiae*, but in the control fermentation process it was as low as 0.487 g g-1 (p<0.05). When the HRT was 12 h the ethanol yield was only 0.365 and 0.318 g g-1, respectively (p<0.05).

There were only few experiments investigating the enhancing ethanol production by ultrasonic stimulation of *S. cerevisiae*. Schläfer et al. (2000) improved biological activity of *S. cerevisiae* by low energy ultrasound assisted bioreactors operated at a frequency of 25 kHz and a power input of 0.3 W L-1. The ethanol production without ultrasonic treatment varied between 3–12 g L-1, while ultrasonic stimulation increased it to 30 g L-1. The highest ethanol concentrations were obtained with a cycle regime of ultrasound exposure and a pause, because during continuous ultrasound irradiation no stimulation in the ethanol fermentation process was recorded.

Lanchun et al. (2003) investigated the influence of low intensity ultrasound on physiological characteristic of *S. cerevisiae*. The results of their study showed, that ultrasounds in the frequency of 24 kHz and the power efficiency of 2 W with 1 s irradiation time every 15 s and 30 min duration cycle, stimulated the material transport and improved the cell's metabolism by changing the osmotic pressure of membrane. Consequently, transfer of substance was speeded up, enzyme synthesis was driven up and enzyme activity was enhanced.

The positive results of the ultrasound treatment on the ethanol production by coimmobilized *S*. *cerevisiae* seemed to be a combination of different processes, including activating the yeast by improving the mass transfer rate of nutrients in the liquid, enhancing the uptake of foreign substances and the release of intracellular products in cells, improving the cell growth and degassing of CO2 (Lanchun et al. 2003; Liu et al., 2007; Liu et al. 2003b). Stimulating enzyme activity is done by increasing in the mass transfer rate of the reagents to the active site (Liu et al., 2007). Ultrasounds irradiation can cause thermal and mechanical stress to biological materials (Liu et al., 2003b). High energy ultrasonic waves break the cells

Feasibility of Bioenergy Production from

utilize by-products of the fermentation process.

fermentation process.

20% (by volume).

**4.1. Materials and methods 4.1.1 Inoculum and wastewater** 

Ultrafiltration Whey Permeate Using the UASB Reactors 211

of hydrogen as a fuel. However, a major doubt on hydrogen as a clean energy alternative is that most of the hydrogen gas is currently generated from fossil fuels by thermochemical processes, such as hydrocarbon reforming, coal gasification and partial oxidation of heavier hydrocarbons (Castellό et al., 2009; Mohan et al., 2007). These methods are considered to be energy intensive and not environmental friendly. It is well known that only biological hydrogen production processes from the fermentation of renewable substrates, such as organic wastewater or other wastes are the promising alternative for hydrogen generation. Several strategies for the production of biohydrogen by fermentation in lab-scale have been found in the literature: photo-fermentation (Gadhamshetty et al., 2008)**,** dark-fermentation (Krupp & Widmann, 2009) and combined-fermentation, which refers to the two fermentations combined (Nath & Das, 2009). However, no strategies for industrial scale productions have been found. In order to define the industrial scale biohydrogen production, more information from laboratory scale experiments are needed, especially related to design and optimization process, and operating parameters. Moreover, generation of biohydrogen by acidogenic phase of anaerobic process (dark-fermentation) is connected with incomplete degradation of organic material into organic acids, so there is a need to

As a result, the fermentative hydrogen production could be coupled with subsequent anaerobic digestion step with the conversion of remaining organic content to biogas. A twostage fermentation process, in which acidogenesis and methanogenesis occur in the separate reactors may offer several advantages, such as improved total wastewater degradation and

The dairy industry produces highly concentrated, carbohydrate-rich wastewaters, but their potential for biohydrogen generation has not been extensively studied. There were some experiences working with cheese whey as the substrate for biohydrogen production (Azbar et al. 2009; Castellό et al., 2009; Venetsaneas et al., 2009). The objectives of this work were: (1) to check the ability to produce biohydrogen using raw, unsterilized UF whey permeate, (2) to combine biohydrogen dark-fermentation process with methane fermentation of biohydrogen production by-products (mainly organic acids) in two-stage continuous

Anaerobic granular sludge from a full-scale UASB reactor treated fruit juice processing wastewater in fruit juice industry, Olsztynek, Poland, was used as an inoculum for biohydrogen and methane production. Prior to inoculation of the hydrogenogenic reactor, the granular sludge was washed with three volumes of tap water and then boiled for 2 h to inactivate hydrogen consuming microorganisms. A final concentration of inoculum was 151 g TSS L-1. No pre-treatment of the granular sludge used for methane production was carried out prior to its inoculation in methanogenic reactor. Initial concentration of inoculum for methane production was 94 g TSS L-1. Both reactors were initially inoculated at a ratio of

UF whey permeate was obtained from a cheese production factory in Nowy Dwór Gdański, Poland. It was received from the factory once a week, was stored at -20°C and was thawed

enhancing biohydrogen and methane production (Venetsaneas et al., 2009).

and denaturate enzymes (Liu et al., 2007; Pitt & Ross, 2003). Low energy ultrasounds can produce a variety of effects on biological materials, including the inhibition or stimulation cellular metabolisms, enzyme activity, alteration of cell membranes and other cellular structures (Liu et al., 2007; Liu et al. 2003a). According to Xie et al. (2008), cavitation is the primary basis of biological effects of low intensity ultrasound. Cavitation bubbles produced by low intensity ultrasound can cause acoustic microstreaming (Xie et al., 2008). The microstreaming surrounding the cells can cause shear stress and enhance the mass transfer, which may stimulate metabolic activities inside the cells (Liu et al., 2003b; Pitt & Ross, 2003; Xie et al., 2008). When ultrasonic intensity is sufficiently low, a stable cavitation occurs and leads to the enhancement of mass transfer and fluid mixing, which produces positive effects on the rate of biological reactions in the exposure systems (Liu et al., 2007).

The growth activity of yeast cells is hardly changed within the early period of sonication regardless of either damage to cell wall, or complete inactivation of the yeast located in the cavitation zone (Tsukamoto et al., 2004). Short sonication time up to 5 min of irradiation indicated bactericidal effects, but the cells were able to repair the damages. According to Guerrero et al. (2005) yeasts, inclusive with *S. cerevisiae*, are highly resistant to ultrasound damage. Moreover, at relatively low intensity of ultrasounds, microorganisms can adapt to the irradiation exposure and their biological activity increases (Liu et al., 2007). With relatively short irradiation period, cell damage and membrane permeability induced by ultrasounds appear to be temporary and reversible. Lanchun et al. (2003) also stated that sonication cannot influence on fermentation strength of *S. cerevisiae* descendants.

### **3.2.3 Conclusions**

The utilization of milk permeate to ethanol in continuous fermentation by co-immobilized *S. cerevisiae* is possible. The optimal ultrasonic intensity and irradiation period are varied in each biological process enhanced by ultrasound and should be find experimentally. According to this experiment, stimulation of yeasts activity could be achieved in the presence of low intensity ultrasound (1 W L-1, 20 kHz), and 1 min every 6 h irradiation period is favorable to increase ethanol production efficiency. Moreover, the short exposure of yeast to ultrasound could reduce the operation costs comparing with continuous irradiation.

For the continuously operating bioreactors, the maximum rates of sugar utilization were 98.9 and 92.4% for the yeast with ultrasound exposure and without ultrasound exposure (p<0.05), respectively*.* The maximum ethanol yield was 0.532 g g-1 lactose, while using *S. cerevisiae* without ultrasound exposure 0.511 g g-1. The study showed that there is no need to extend the HRT over 36 h or more, because most of the lactose was converted into ethanol during 24 h (95.6% in the ultrasound-assisted fermentation).

All results obtained here raises the new perspectives for disposal UF whey permeate.
